Circular RNA circFGFR1 Functions as an Oncogene in Glioblastoma Cells through Sponging to hsa-miR-224-5p

Recently, increased studies have shown the important regulatory role of circular RNA (circRNA) in cancer progression and development, including glioblastoma (GBM). However, the function of circRNAs in glioblastoma is still largely unclear. Here, we state that circFGFR1 is elevated in glioma cells, resulting in aggravated glioma aggravated malignancy. The upregulation of circFGFR1 also promotes glioma growth in mouse xenograft models. Furthermore, CXCR4 level in glioma cells is positively correlated with circFGFR1 level, and higher CXCR4 expression is found in circFGFR1 overexpression groups. The effect of circFGFR1 on glioma malignancy is abolished in CXCR4 knockout cells. Then, RIP, RNA pull-down, and luciferase reporter assay results showed that hsa-miR-224-5p directly binds to circFGFR1 and CXCR4 mRNA. The CXCR4 3 ′ -untranslated region (UTR) activated luciferase activity was reduced with hsa-miR-224-5p transfection, while it is reversed when cotransfected with circFGFR1, indicating that circFGFR1 acts as a hsa-miR-244-5p sponge to increase CXCR4 expression. The hsa-miR-224-5p expression is negatively corrected with the glioma malignancy through inhibiting CXCR4 level. Besides, the circFGFR1-induced regulation in glioma malignancy is also abrogated in hsa-miR-224-5p knockout cells. Taken together, our findings suggest that circFGFR1 plays a critical role in the tumorigenic behaviors in glioma cells by upregulating CXCR4 expression via sponging to hsa-miR-224-5p. These findings provide a new perspective on circRNAs during GBM development.

Association of CXCR4 mRNA Expression with Clinicopathological Aspects of Invasive Breast Carcinoma

<p class="Abstract">ABSTRACT</p><p><strong> </strong></p><p><strong>Background</strong></p><p>Breast cancer is the most common malignancy in women of which majority histological type is Invasive (Ductal) Carcinoma of No Special Type (NST). The prognosis in breast carcinoma is influenced by many factors such as age, tumor size, degree of histology, and lymph node metastasis. Another factor in the development and metastasis of breast cancer is the chemokine receptor CXCR4 and its ligand, CXCL12. Studies state that the expression of CXCR4 in Breast Invasive Carcinoma associated with clinicopathologic aspects remain unclear. This study aims to determine differences in the level of CXCR4 mRNA expression between clinicopathologic aspects in breast carcinoma..</p><p><strong>Method</strong></p><p>A total of 50 samples of formalin-fixed paraffin-embedded (FFPE) tissues diagnosed as invasive breast carcinoma (NST) are used in this study. Samples are divided into groups, namely with and without lymph node metastasis, age &lt;45 years and&gt; 45 years, small and large size, low grade and high grade. CXCR4 mRNA expression is quantitatively examined by qRT-PCR. CXCR4 mRNA expression differences between various clinicopathologic aspects were analyzed by One-Way ANOVA</p><p><strong>Result</strong></p><p>Of the 50 samples, 26 samples (52%) revealed increased expression of CXCR4 mRNA compared to normal tissue. There were no significant differences in mRNA expression of CXCR4 between various prognostic factors (p&gt; 0.05) such as the status of lymph node metastasis, histologic grading, size, and age. However, the expression of CXCR4 mRNA is increased in breast carcinoma when compared to normal breast tissue. Nonetheless the level of CXCR4 expression alone is not associated to clinicopathologic aspects in invasive breast carcinoma.</p><p><strong>Conclusion</strong></p><p>CXCR4 mRNA expression did not differ significantly between the various clinicopathological aspects of invasive breast carcinoma.</p><p> </p><p><strong><em>Keyword</em></strong><strong>: </strong>invasive breast carcinoma, mRNA of CXCR4, Clinicopathologic aspects</p><p> </p>

CXCR4 expression in glioblastoma tissue and the potential for PET imaging and treatment with [68Ga]Ga-Pentixafor /[177Lu]Lu-Pentixather

Purpose CXCR4 (over)expression is found in multiple human cancer types, while expression is low or absent in healthy tissue. In glioblastoma it is associated with a poor prognosis and more extensive infiltrative phenotype. CXCR4 can be targeted by the diagnostic PET agent [68Ga]Ga-Pentixafor and its therapeutic counterpart [177Lu]Lu-Pentixather. We aimed to investigate the expression of CXCR4 in glioblastoma tissue to further examine the potential of these PET agents. Methods CXCR4 mRNA expression was examined using the R2 genomics platform. Glioblastoma tissue cores were stained for CXCR4. CXCR4 staining in tumor cells was scored. Stained tissue components (cytoplasm and/or nuclei of the tumor cells and blood vessels) were documented. Clinical characteristics and information on IDH and MGMT promoter methylation status were collected. Seven pilot patients with recurrent glioblastoma underwent [68Ga]Ga-Pentixafor PET; residual resected tissue was stained for CXCR4. Results Two large mRNA datasets (N = 284; N = 540) were assesed. Of the 191 glioblastomas, 426 cores were analyzed using immunohistochemistry. Seventy-eight cores (23 tumors) were CXCR4 negative, while 18 cores (5 tumors) had both strong and extensive staining. The remaining 330 cores (163 tumors) showed a large inter- and intra-tumor variation for CXCR4 expression; also seen in the resected tissue of the seven pilot patients—not directly translatable to [68Ga]Ga-Pentixafor PET results. Both mRNA and immunohistochemical analysis showed CXCR4 negative normal brain tissue and no significant correlation between CXCR4 expression and IDH or MGMT status or survival. Conclusion Using immunohistochemistry, high CXCR4 expression was found in a subset of glioblastomas as well as a large inter- and intra-tumor variation. Caution should be exercised in directly translating ex vivo CXCR4 expression to PET agent uptake. However, when high CXCR4 expression can be identified with [68Ga]Ga-Pentixafor, these patients might be good candidates for targeted radionuclide therapy with [177Lu]Lu-Pentixather in the future.

Biphasic MIF and SDF1 expression during podocyte injury promote CD44-mediated glomerular parietal cell migration in focal segmental glomerulosclerosis

Glomerular parietal epithelial cell (PEC) activation, as revealed by de novo expression of CD44 and cell migration toward the injured filtration barrier, is a hallmark of podocyte injury-driven focal segmental glomerulosclerosis (FSGS). However, the signaling pathway that mediates activation of PECs in response to podocyte injury is unknown. The present study focused on CD44 signaling, particularly the roles of two CD44-related chemokines, migration inhibitory factor (MIF) and stromal cell-derived factor 1 (SDF1), and their common receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), in the NEP25/LMB2 mouse podocyte-toxin model of FSGS. In the early phase of the disease, CD44-positive PECs were locally evident on the opposite side of the intact glomerular tuft and subsequently increased in the vicinity of synechiae with podocyte loss. Expression of MIF and SDF1 was first increased in injured podocytes and subsequently transferred to activated PECs expressing CD44 and CXCR4. In an immortalized mouse PEC (mPEC) line, recombinant MIF and SDF1 (rMIF and rSDF1, respectively) individually increased CD44 and CXCR4 mRNA and protein levels. rMIF and rSDF1 stimulated endogenous MIF and SDF1 production. rMIF- and rSDF1-induced mPEC migration was suppressed by CD44 siRNA. However, MIF and SDF1 inhibitors failed to show any impact on proteinuria, podocyte number, and CD44 expression in NEP25/LMB2 mice. Our data suggest that injured podocytes upregulate MIF and SDF1 that stimulate CD44 expression and CD44-mediated migration, which is enhanced by endogenous MIF and SDF1 in PECs. This biphasic expression pattern of the chemokine-CD44 axis in podocytes and PECs may be a novel mechanism of “podocyte-PEC cross-talk” signaling underlying podocyte injury-driven FSGS.

Predicting Angiogenesis by Endothelial Progenitor Cells Relying on In-Vitro Function Assays and VEGFR-2 Expression Levels

Clinical trials have demonstrated the safety and efficacy of autologous endothelial progenitor cell (EPC) therapy in various diseases. Since EPCs’ functions are influenced by genetic, systemic and environmental factors, the therapeutic potential of each individual EPCs is unknown and may affect treatment outcome. Therefore, our aim was to compare EPCs function among healthy donors in order to predict blood vessel formation (angiogenesis) before autologous EPC transplantation. Human EPCs were isolated from the blood of ten volunteers. EPCs proliferation rate, chemoattractant ability, and CXCR4 mRNA levels were different among donors (p < 0.0001, p < 0.01, p < 0.001, respectively). A positive correlation was found between SDF-1, CXCR4, and EPCs proliferation (R = 0.736, p < 0.05 and R = 0.8, p < 0.01, respectively). In-vivo, blood vessels were counted ten days after EPCs transplantation in a subcutaneous mouse model. Mean vessel density was different among donors (p = 0.0001); nevertheless, donors with the lowest vessel densities were higher compared to control (p < 0.05). Finally, using a linear regression model, a mathematical equation was generated to predict blood vessel density relying on: (i) EPCs chemoattractivity, and (ii) VEGFR-2 mRNA levels. Results reveal differences in EPCs functions among healthy individuals, emphasizing the need for a potency assay to pave the way for standardized research and clinical use of human EPCs.

Effects of miR-338 on morphine tolerance by targeting CXCR4 in a rat model of bone cancer pain

The present study aimed to investigate the effects of miR-338 on morphine tolerance through the targeting of CXC chemokine receptor-4 (CXCR4) in a rat model of bone cancer pain (BCP). Sprague–Dawley (SD) rats were obtained and divided into model saline (n=10), model morphine (n=50), normal saline (n=10) and normal morphine (healthy rats, n=10) groups. After BCP rat model establishment, the remaining SD rats (n=40) in the model saline group were assigned into pLV-THM-miR-338, pLV-THM-anti-miR-338, CXCR4 shRNA, blank and PBS groups. Luciferase reporter gene assay was used for luciferase activity. Quantitative real-time PCR (qRT-PCR) and Western blotting were performed to detect the miR-338 and CXCR4 mRNA and protein expression. The model saline group showed increased mRNA and protein expressions of CXCR4 but decreased miR-338 compared with the model saline group, and the model morphine group had increased mRNA and protein expressions of CXCR4 but decreased miR-338 compared with the model saline group. The mRNA and protein expressions of miR-338 in the pLV-THM-miR-338 group increased remarkably while those of the pLV-THM-anti-miR-338 group decreased significantly compared with the CXCR4 shRNA, blank and PBS groups. The pLV-THM-miR-338, pLV-THM-anti-miR-338, CXCR4 shRNA and CXCR4 mRNA groups all had lower mRNA and protein expressions of CXCR4 than those in the blank and PBS groups. miR-338 exerts significant influence in the inhibition of morphine tolerance by suppressing CXCR4 in BCP.

Inhibition of HDAC5-Mediated p65 Deacetylation Enhances Human Cord Blood Hematopoietic Stem Cell Homing and Engraftment in NSG Mice

Stromal cell derived factor-1α (SDF-1α)/chemokine C-X-C receptor 4 (CXCR4) interactions play a crucial role in hematopoietic stem cells (HSC) trafficking and homing to the bone marrow (BM) environment. To identify new epigenetic regulators of CXCR4 receptor, we screened a chemical compound library of epigenetic enzyme inhibitors to evaluate their effects on membrane CXCR4 expression in CB CD34+ cells. We found that treatment with a couple of histone deacetylase (HDAC) inhibitors, including M344, strongly upregulated membrane CXCR4 expression. We also investigated the effect of M344 on membranal CXCR4 expression in a rigorously defined more primitive HSC cell population (CD34+CD38-CD45RA-CD49f+CD90+) and found that there was a 2.5 fold increase in the M344 treated group compared with vehicle control treated cells. Quantitative RTPCR also showed increased CXCR4 mRNA levels in M344-treated CD34+ cells compared with vehicle control (3.1 fold), indicating that this regulation occurs at the transcriptional level. We next evaluated the effect of M344 treatment on HSC chemotaxis in in vitro transwell migration assays. Both vehicle and M344 treated CB CD34+ cells showed significant migration to 50ng/mL SDF-1α, however, chemotaxis was 2.1 fold higher in M344 treated group. Enhanced migration to SDF-1α by M344 was also observed in the more primitive HSC population. Chemotaxis of CB CD34+ cells to SDF-1α was blocked by CXCR4 antagonist AMD3100, suggesting that the effect was mediated through the CXCR4 receptor. To directly evaluate in vivo homing, vehicle and M344 treated CB CD34+ cells were injected into sublethally irradiated NSG mice, and human cells homing to mouse BM were analyzed 24 hours after transplantation. Consistently, M344 treatment enhanced CB CD34+ cells homing by 2.3 fold in NSG mice. Next, we performed a limiting dilution assay to compare the frequency of SCID-repopulating cells (SRCs) in vehicle and M344 treated CB CD34+ cells. Poisson distribution analysis revealed an SRC frequency of 1/3216 in vehicle control treated group and 1/746 in M344 treatment. We calculated the respective presence of 310.9 SRCs and 1340.5 SRCs in 1×106 cells from vehicle control and M344-treated cultures, so M344 treatment resulted in a 4.3 fold increase in the number of functionally detectable SRCs compared with vehicle control. Eighteen HDACs have been identified in humans and they are divided into four classes. The mechanisms regarding HDAC regulation of HSC homing and engraftment are largely unknown. Using shRNA to knockdown expression of individual HDACs in CB CD34+ cells, we surprisingly found that HDAC5 shRNA transfection resulted in upregulation of membrane CXCR4 expression. LMK235, a selective inhibitor of HDAC5, also increased membrane CXCR4 expression in CB CD34+ cells. In contrast, inhibition of other HDACs did not show any effect on membrane CXCR4 expression. Similar to M344, LMK235 treatment resulted in significantly higher CXCR4 mRNA, membrane CXCR4 expression in CB HSCs, enhanced migration to SDF-1α in chemotaxis assay, and higher number of cells homed to the BM in NSG mice. We next performed chromatin immunoprecipitation (ChIP) assays to examine the chromatin status at the CXCR4 promoter region. H3K9 acetylation levels were significant higher in LMK235-treated CB CD34+ cells compared with vehicle control, suggesting increased H3K9 levels at the CXCR4 promoter region contributes to increased CXCR4 transcription. To further explore the mechanisms underlying HDAC5 regulation of HSC homing, we tested a couple of signaling pathways and found that inhibition of the NF-κB signaling pathway by Andrographolide and Pyrrolidinedithiocarbamate Ammonium, suppressed M344 and LMK235-mediated CXCR4 upregulation on CB CD34+ cells. It has been reported that acetylation of NF-κB p65 subunit enhances its transcriptional activity. We examined the acetylation levels of p65 and found that LMK235 treatment resulted in increased levels of p65 acetylation in CB CD34+ cells, indicating p65 could be a downstream target of HDAC5. Consistently, using ChIP assay we detected increased levels of acetylated p65 binding to the CXCR4 promoter region in the LMK235-treated group. Taken together, our results reveal previously unknown specific epigenetic regulation of HSC homing and engraftment by HDAC5, which suggests a new translational strategy to enhance HSC transplantation. Disclosures Broxmeyer: CordUse: Other: SAB Member.

Extracts fromCurcuma zedoariaInhibit Proliferation of Human Breast Cancer Cell MDA-MB-231In Vitro

Objective.To evaluate the effect of petroleum ether extracts ofCurcuma zedoariaon the proliferation of human triple negative breast cancer cell line MDA-MB-231.Methods.The reagents were isolated fromCurcuma zedoariaby petroleum ether fraction. It was assayed by CCK8 for MDA-MB-231 cellular viability with various concentrations and days, cell cycle analyses, Western Blot analysis, and Realtime Reverse Transcriptase PCR analyses for chemokines molecules including E-cadherin, and E-selectin, and adhesion molecules including CCR7, SLC, SDF-1, and CXCR4. Epirubicin was used as control in the study.Results.MDA-MB-231 cells were inhibited by petroleum ether extracts ofCurcuma zedoaria(P < 0.05), and the inhibition rate was dependent on concentrations and time. Petroleum ether extracts ofCurcuma zedoariaas well as Epirubicin produce a significant G0/G1 cell cycle arrest. The level of expression of proteins E-cadherin and E-cadherin mRNA was significantly increased, while proteins SDF-1, CCR7, and CXCR4 mRNA were decreased after being incubated with petroleum ether extracts ofCurcuma zedoariaat the concentrations of 300 μg/mL than control (P < 0.05). The differences were that the protein CXCR4 mRNA expression level was higher than vehicle.Conclusions.MDA-MB-231 cells were inhibited by petroleum ether extracts ofCurcuma zedoaria.